
==== Front
iScience
iScience
iScience
2589-0042
Elsevier

S2589-0042(24)01956-4
10.1016/j.isci.2024.110731
110731
Article
Persistent Mycobacterium tuberculosis bioaerosol release in a tuberculosis-endemic setting
Dinkele Ryan 12
Gessner Sophia 12
Patterson Benjamin 3
McKerry Andrea 4
Hoosen Zeenat 4
Vazi Andiswa 4
Seldon Ronnett 4
Koch Anastasia 12
Warner Digby F. digby.warner@uct.ac.za
125∗
Wood Robin robin.wood@hiv-research.org.za
24∗∗
1 UCT Molecular Mycobacteriology Research Unit, Department of Pathology, Faculty of Health Sciences, University of Cape Town, Cape Town 7925, South Africa
2 Institute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town 7925, South Africa
3 Amsterdam Institute for Global Health and Development, University of Amsterdam, Amsterdam 1105, the Netherlands
4 Aerobiology and TB Research Unit, Desmond Tutu Health Foundation, Cape Town 7925, South Africa
5 Wellcome Centre for Infectious Diseases Research in Africa, Faculty of Health Sciences, University of Cape Town, Cape Town 7925, South Africa
∗ Corresponding author digby.warner@uct.ac.za
∗∗ Corresponding author robin.wood@hiv-research.org.za
20 8 2024
20 9 2024
20 8 2024
27 9 11073121 5 2024
22 7 2024
12 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Summary

Pioneering studies linking symptomatic disease and cough-mediated Mycobacterium tuberculosis (Mtb) release established the infectious origin of tuberculosis (TB), simultaneously informing the notion that pathology is a prerequisite for Mtb transmission. Our recent work has challenged this assumption: by sampling TB clinic attendees, we detected equivalent release of Mtb-containing bioaerosols by confirmed TB patients and individuals not receiving a TB diagnosis and observed time-dependent reduction in Mtb bioaerosol positivity during 6-month follow-up of both cohorts, irrespective of anti-TB chemotherapy. Now, we report widespread Mtb release in our TB-endemic setting: of 89 randomly recruited community members, 79.8% (71/89) produced Mtb-containing bioaerosols independently of QuantiFERON status, a standard test for Mtb exposure. Moreover, during 2-month longitudinal sampling, only 2% (1/50) were serially Mtb bioaerosol negative. These results necessitate a reframing of the prevailing paradigm of Mtb transmission and TB etiology, perhaps explaining the historical inability to elucidate Mtb transmission networks in TB-endemic regions.

Graphical abstract

Highlights

• Pathology is widely considered a prerequisite for M. tuberculosis (Mtb) transmission

• Subclinical TB is common and increasingly recognized as a driver of Mtb transmission

• The contribution of asymptomatic infection to Mtb transmission remains unknown

• Aerosolization of Mtb is common in high-TB regions and undetectable by standard tests

Microbiology

Subject areas

Microbiology
Published: August 20, 2024
==== Body
pmcIntroduction

Infection with Mycobacterium tuberculosis (Mtb) encompasses a spectrum of outcomes.1,2,3,4 At its mildest, the immune system clears or contains infecting Mtb bacilli with minimal discernible impact on the host.5 At its most severe, pulmonary Mtb infection results in advanced disease that is associated with a 40%–70% fatality rate in the absence of effective treatment.6 Despite the rarity of progression to active disease at an individual level and the availability of effective chemotherapy,5,7 tuberculosis (TB) remains a leading global cause of mortality,8 with notification rates surpassing those reported in the early 1900s in certain settings.6

In endemic regions, most incident TB is thought to arise from the recent (<2 years) transmission of Mtb.9,10 However, with only 1%–30% of new Mtb infections traceable to known TB cases,11,12,13,14 it is likely that numerous undetected sources of Mtb transmission exist within these communities. Consistent with this possibility, there is growing interest in the notion that early TB states—encompassing Mtb infection, subclinical, and clinical TB4—might provide a previously overlooked reservoir of transmission.15,16 Establishing the propensity for Mtb release—often equated with “infectiousness”—during early TB is therefore essential but very difficult to accomplish.17

Sputum diagnosis is generally considered the gold standard for assessing infectiousness among TB patients.18 Through this lens, data from extensive community TB screening efforts utilizing sputum-GeneXpert—which have identified large numbers of asymptomatic cases19—support the notion of unrecognized transmitters in high-burden communities. And, although these results accommodate the potential for Mtb transmission to occur prior to the development of recognizable TB symptoms,16 they nevertheless rely on sputum production (itself a symptom of disease) for the detection of Mtb. This dependency on sputum production imposes a significant constraint on attempts to detect individuals with early TB and is exacerbated by the difficulties encountered in obtaining useable samples from many individuals.18

Bioaerosol sampling offers a non-invasive method to collect peripheral lung fluid and/or particulate matter independent of the specific disease state.20 Moreover, the detection of Mtb in bioaerosols signifies the release of bacilli by an individual, tentatively offering a concurrent metric of transmission risk.21,22 Despite these advantages, the utilization of bioaerosol sampling has predominantly been restricted to individuals with bacteriologically confirmed TB. This owes mainly to the challenges inherent in handling extremely paucibacillary samples as well as the assumed importance of frank pathology for Mtb release—in turn reinforcing the primacy of sputum bacterial burden as key predictor of infectiousness. Insights gained from the COVID-19 pandemic, however, established the plausibility of asymptomatic transmission,23,24 with analogous recent results suggesting the cough-independent aerosolization of Mtb.25,26 It seems possible, therefore, that individuals might progress from Mtb infected (often inferred via interferon gamma release assay [IGRA],27 of which the QuantiFERON-TB Gold [QFT] test is the predominant version) to infectious before producing sputum.

Since 2013, we have studied the spontaneous generation of Mtb-containing bioaerosols using the respiratory aerosol sampling chamber (RASC), a highly sensitive, adaptable personal clean room designed to capture all particulate matter (including Mtb bacilli) released by individuals.28 By combining liquid capture of bioaerosols in the RASC with DMN-trehalose-enabled microscopic detection and visualization of Mtb bacilli, we recently reported that ∼90% of clinic attendees with presumptive TB produced bioaerosols containing viable Mtb bacilli.29 Surprisingly, the high proportion of Mtb bioaerosol positivity was independent of final TB diagnosis: Mtb bioaerosol release was common to both notified TB patients—who commenced anti-TB chemotherapy—and those not diagnosed with TB (who did not initiate treatment). Moreover, during 6-month follow-up, the rates of decline in symptom severity and Mtb bioaerosol release were equivalent in both groups. And, at the 6-month study endpoint, approximately 20% of all participants—including treated TB patients and those who did not receive a TB diagnosis—remained Mtb bioaerosol-positive despite clinical resolution, consistent with prior observations from radiological30 and other assays.31

Given the high prevalence of Mtb bioaerosol release among clinic attendees not diagnosed with TB, as well as the frequency of Mtb bioaerosol release in confirmed TB patients on completion of standard anti-TB treatment, we hypothesized that the production of Mtb bioaerosols by individuals living in TB-endemic communities might be more common than previously thought. To investigate this possibility, we utilized our bioaerosol sampling platform25 to investigate Mtb release in 89 randomly selected participants recruited into two consecutive cohorts: the first was a cross-sectional community survey comprising 39 participants, and the second was a longitudinal observational study in which 50 individuals consented to be serially sampled at three separate time points over 2 months. As detailed below, our results suggest that early-stage Mtb infection is pervasive and may be an important contributor to the transmission of Mtb in high TB-burden communities.

Results

High-prevalence Mtb bioaerosol release in a TB-endemic community independent of respiratory maneuver

To investigate the prevalence of Mtb bioaerosol release in a TB-endemic setting, we initiated a cross-sectional community survey in Masiphumelele, Cape Town, with recruitment randomized geospatially by parcel of land or “erf” (Figure 1). From 10 erfs, we sequentially enrolled 39 individuals (1–12 participants/erf [median = 3]) who were assessed in Mtb bioaerosol release, sputum-GeneXpert MTB/RIF (GXP), and QuantiFERON-TB Gold (QFT) assays. The cohort had high rates of human immunodeficiency virus (HIV) infection (13%) and previous TB (18%). Two participants who produced Mtb bioaerosols at the screening visit (5%) were GXP-positive at follow-up (one of these participants had completed treatment for TB in 2020, 2 years prior to the study), providing immediate evidence of undiagnosed TB in the community (Table 1).Figure 1 Participant recruitment and bioaerosol sampling algorithms for the two randomized community cohorts

(A) For the initial cross-sectional survey, 39 participants were recruited from randomly selected erfs in Masiphumelele. At a first screening visit, participants produced bioaerosol samples from three respiratory maneuvers: forced vital capacity (FVC), tidal breathing (TiBr), and induced cough. Samples were processed and visualized independently by microscopists blinded to all sample information. Owing to the high prevalence of Mtb bioaerosol positivity, all participants were brought back for a follow-up visit during which blood and sputum were collected for QFT and GXP analysis, respectively.

(B) For the longitudinal study of Mtb bioaerosol release, 50 participants were recruited. Blood and sputum samples were collected at baseline for QFT and GXP analyses, respectively. Two equivalent bioaerosol samples were collected during 10 min of tidal breathing with deep breaths taken at 30-s intervals. These samples were processed and imaged independently on nanowell-arrayed microscope slides by microscopists blinded to all sample information. This process was repeated at 2 weeks and 2 months after initial recruitment.

Table 1 Summary of participant demographic and clinical information from the two cohorts

Variable	Category	Cohort 1	Cohort 2	p value	
n (%)	n (%)	
Total		39 (100)	50 (100)	–	
Gender	Male	12 (30.8)	13 (26)	0.64	
Previous TB	Yes	7 (17.9)	4 (8)	0.20	
HIV status	Negative	29 (74.4)	38 (76)	0.02	
	Positive	5 (12.8)	12 (24)		
	Unknown	5 (12.8)	0 (0)		
GeneXpert Ultra	Negative	34 (87.2)	48 (96)	>0.99	
	Positive	2 (5.1)	2 (4)		
	NA	3 (7.7)	0 (0)		
QuantiFERON-TB Gold	Negative	17 (43.6)	16 (32)	0.15	
	Positive	16 (41)	33 (66)		
	Indeterminate	2 (5.1)	1 (2)		
	NA	4 (10.3)	0 (0)		
Age [mean (sd)]		32.9 (12.6)	34.2 (12.5)	0.636	
NA = not available (incomplete or failed assay); Excluded from the analysis.

Advanced lung pathology and chronic cough are often viewed as essential for Mtb release.32 This assumption is, however, incompatible with the notion that Mtb may transmit during early-stage infection. Here, we examined the potential for Mtb release from a randomly selected community cohort, considering both unrelated cough33 and tidal breathing25 as potential mechanisms. This was done by comparing Mtb aerosolization from three independently sampled respiratory maneuvers, namely, forced vital capacity (FVC—a deep breathing respiratory maneuver), tidal breathing (TiBr), and cough, according to our previously described methodology.25

When comparing the respiratory maneuvers, the percentage of Mtb-positive samples ranged from 51.1% to 67.6%, with no significant differences observed in the likelihood of producing Mtb between the three respiratory maneuvers (Figures 2A and 2B). Moreover, no differences were observed in the average number of bacteria identified per sample (Figures 2C and 2D). Across FVC, TiBr, and cough, the average counts were 2.9 (95% confidence interval [CI]: 1.9; 4.0), 2.2 (95% CI: 1.4; 3.2), and 2.0 (95% CI: 1.1; 3.1) Mtb bacilli per sample, respectively. Overall, we found that 79.5% (31/39) of the participants produced at least one positive bioaerosol sample when considering all three maneuvers. Together, these data suggest that the prevalence of Mtb release is high in a TB-endemic setting.Figure 2 High-prevalence Mtb bioaerosol release in a TB-endemic community independent of respiratory maneuver

(A) The percentage of samples in which putative Mtb were detected (turquoise) or absent (purple) from forced vital capacity [FVC (67.6%)], tidal breathing [TiBr (51.4%)], and cough (51.1%).

(B) Results of a logistic regression comparing the odds of a positive bioaerosol result compared to TiBr.

(C) Box and whisker and equivalent density plots comparing the total number of Mtb detected between the three respiratory maneuvers. White circle and error bars overlayed onto the box and whisker plots represent the mean ±95% CI.

(D) Results of a negative binomial regression comparing the number of Mtb detected between the three respiratory maneuvers. OR = odds ratio, IRR = incident rate ratio, CI = confidence interval, BA = bioaerosol.

Altering the bioaerosol sampling algorithm did not reduce Mtb detection efficiency

The observation that ∼80% of a randomly selected community cohort produced Mtb bioaerosols was reminiscent of our previous work, which demonstrated the high baseline prevalence (∼90%) of Mtb release among TB clinic attendees with presumptive disease, regardless of final diagnosis or respiratory maneuver.29 However, the cross-sectional community survey only provided a snapshot in time of Mtb bioaerosol release. To investigate the variability of Mtb release through time, we repeated our random community sampling strategy in recruiting a further 50 individuals from 20 erfs (1–7 participants/erf [median = 2]) into a longitudinal observational study of Mtb release (Figure 1).

Bioaerosols were collected at baseline, and follow-up visits were scheduled at 2 weeks and 2 months postbaseline, in accordance with previously described time intervals.29 The characteristics of this cohort were not significantly dissimilar to the first 39 participants (Table 1), except for HIV status—where the difference was driven largely by the proportion of individuals reporting their status as “unknown.” Given the observation from the initial cohort that FVC and TiBr were sufficient to aerosolize Mtb, we reasoned our bioaerosol sampling algorithm could be simplified by removing induced cough. Therefore, rather than collecting three 5-min samples (∼15 min), each from a different respiratory maneuver, we implemented a single sampling algorithm, which was repeated twice: 10 min of tidal breathing with deep breaths every 30 s (∼20 min sampling in total). This enabled head-to-head comparison of duplicate bioaerosol samples at each visit, allowing for an assessment of variation through time.

We found comparable results between first and second cohorts in both the proportion of participants producing at least one positive sample and the total number of aerosolized Mtb (Figures 3A–3C). Average Mtb counts at baseline varied from 0 to 16, with 20% (10/50) of the participants producing two negative bioaerosol samples (Figure 4A). Discrepant samples (i.e., where one sample was negative and the other positive) were infrequent [22% (11/50)] with a low maximum sample difference (three bacilli). There was a strong correlation between the first and second bioaerosol sample (Figure 4B) and a high degree of agreement, with 60% of the samples differing by ± 1 (Figures 4C and 4D). Considering the relatively small sample size of each cohort and the comparability of the bioaerosol results, we pooled the data to investigate which covariates were associated with Mtb release. No covariates were associated with increased odds of an Mtb-positive sample (Table 2). In contrast, biological sex (incident rate ratio [IRR] = 1.88, p < 0.05), previous TB (IRR = 2.33, p < 0.05), and age ≥45 years (IRR = 0.48, p < 0.05) were associated with the number of Mtb bacilli detected (Table 3). Surprisingly, neither QFT status—the conventional marker of Mtb infection—nor HIV status was associated with Mtb release. Overall, these data suggest that individuals who aerosolize Mtb do so consistently within short time frames and independently of standard markers of infection.Figure 3 Altering the bioaerosol sampling algorithm did not reduce Mtb detection efficiency

(A) The percentage of samples in which putative Mtb were detected (green) or absent (purple). The odds of a positive bioaerosol sample were equivalent between the two groups (OR = 1.03, 95% CI = 0.36; 2.92, p = 0.952) (B) Box and whisker and equivalent density plots comparing the total number of Mtb detected between the two cohorts. The rates at which Mtb were produced during the two samplings were equivalent (IRR = 0.797, 95% CI = 0.47; 1.33, p = 0.386). White circle and error bars overlayed onto the box and whisker plots represent the mean ±95% CI. OR = odds ratio, IRR = incident rate ratio, CI = confidence interval, BA = bioaerosol.

Figure 4 The consistent production of aerosolized Mtb during two equivalent respiratory maneuvers

(A) Plot of the mean Mtb (DMN-tre positive) count from two samples, with error bars representing the range. No lines indicate equal counts, green lines indicate one count = 0, blue lines indicate two counts >0.

(B) Plot of the Mtb (DMN-tre positive) counts of the first and second samples at baseline (r = 0.810, p < 0.0001) with a fitted line representing a 1:1 correlation.

(C) A Bland-Altman plot indicating the level of agreement between the first and second samples, with a (D) histogram showing the frequency of each count difference. Most samples differed by either 0 or ±1 (60%) and 94% of the samples differed by four or less.

Table 2 Results of a logistic regression assessing the odds of a positive bioaerosol sample

Variable	Category	OR	95% CI	p value	
Age	≥45	1.52	0.34; 10.8	0.622	
Biological sex	Male	1.44	0.38; 7.14	0.619	
Previous TB	Yes	1.12	0.24; 8.13	0.898	
QFT golda	Positive	1.44	0.44; 4.66	0.543	
HIV statusb	Positive	0.63	0.17; 2.66	0.495	
OR = odds ratio, CI = confidence interval.

a Indeterminate results removed.

b Unknown status removed.

Table 3 Results of a negative binomial regression assessing the number of Mtb per participant

Variable	Category	IRR	95% CI	p value	
Age	≥45	0.48	0.24; 1.03	0.039	
Biological sex	Male	1.88	1.07; 3.42	0.029	
Previous TB	Yes	2.33	1.18; 5.02	0.015	
QFT golda	Positive	1.01	0.60; 1.68	0.958	
HIV statusb	Positive	0.86	0.45; 1.70	0.649	
IRR = incident rate ratio, CI = confidence interval.

a Indeterminate results removed.

b Unknown status removed.

Persistent Mtb release among a randomly selected community cohort is common

Based on the observation from baseline sampling that 94% of matched samples differed by fewer than five Mtb bacilli, we reasoned that differences in the average count between two consecutive samples through time of five or greater could be considered significant. According to this definition, most (74% [32/43]) participants were constant low-level producers of Mtb over 2 months (Figure 5A). The rate of bioaerosol positivity was relatively consistent through time, ranging from 76.7% to 90.7% (Figures 5B and 5C). Most surprisingly, only one individual was negative across all three visits (representing six negative bioaerosol samples in total). Of 43 individuals with complete data for all three time points (baseline, 2 weeks, 2 months), 19% (8/43) and 9% (4/43) were negative at one and two visits, respectively. Although individual participants varied in their bioaerosol count from visit to visit, there was no overall trend in the number of Mtb detected per visit (Figures 5D and 5E). This lack of secular trend in Mtb bioaerosol release among a predominantly asymptomatic, randomly selected community cohort supports our previous hypothesis that symptomatic Mtb bioaerosol clearance is immune driven.29Figure 5 Persistent Mtb release among a randomly selected community cohort is common

(A) Total Mtb (DMN-tre positive) counts (sum of the two samples) through time, stratified by time trend.

(B) The percentage of samples in which putative Mtb were detected (turquoise) or absent (purple) at each of the visits.

(C) Results of a logistic regression comparing the odds of a negative BA result compared to T0.

(D) Box and whisker and density plots comparing the total number of Mtb bacilli (DMN-tre positive) detected at each visit. White circle and error bars overlayed onto the box and whisker plots represent the mean ±95% CI.

(E) Results of a negative binomial regression comparing the number of Mtb bacilli (DMN-tre positive) detected at each visit. OR = odds ratio, IRR = incident rate ratio, CI = confidence interval, BA = bioaerosol.

Discussion

The use of new tools in TB research has enabled unexpected insights that question established models of pathogenicity. For Mtb transmission, the increasing awareness that TB disease is not required for Mtb bioaerosol release interrogates previous assumptions linking pathology to infectiousness,17 in turn raising important questions about the implications for new diagnostics, drugs, and vaccines.34

In work immediately preceding this report, we detected high-prevalence Mtb bioaerosol release among TB clinic attendees, irrespective of diagnosis. Moreover, persistent release of Mtb bioaerosols was observed post-treatment among bacteriologically confirmed TB cases,29 a finding consistent with separate work that used positron emission tomography–computed tomography (PET-CT) imaging and Mtb mRNA detection to conclude that apparently curative treatment for TB might not eradicate all Mtb bacteria.30 One implication of our previous results was that release of Mtb bioaerosols within TB-endemic communities might be considerably more prevalent than previously understood. To address that possibility, we initiated the work reported here, in which we broadened our sampled population to screen a random selection of individuals from the larger of the two communities served in the earlier study. In doing so, we modified the sampling protocol slightly to exclude forced coughing and, importantly, demonstrated high reproducibility in Mtb capture numbers when repeated samples were obtained from the same individual during the same visit.

The results reported here were again unexpected: aerosolized Mtb was detectable in 80% of participants recruited at random in our high TB-burden community. Moreover, Mtb release occurred independently of QFT results, the standard test for current or previous exposure to Mtb.27 From longitudinal sampling of 50 participants over 2 months, it was apparent that most individuals were transiently or persistently releasing Mtb: only one participant returned negative Mtb-bioaerosol results across all three visits (representing six negative bioaerosol samples). Despite variation in Mtb release per participant, no overall trend was observed in Mtb release through time for the cohort. This observation contrasts with our previous report of the spontaneous clearance of Mtb bioaerosols by predominantly symptomatic clinic attendees with presumptive TB.29 It may be that, in apparently healthy individuals, the containment of infection is achieved independently of an interferon gamma response or through innate immunity. However, it is tempting to speculate that, in most people, Mtb bacilli are able to avoid immune surveillance or are in a relatively immune-privileged site—a possibility strengthened by several recent studies suggesting the ability of Mtb to avoid exposure to immune surveillance,35,36 perhaps through occupation of intercellular niches.37 Determining the immune correlates of protection and/or the anatomical location of bacilli released in bioaerosols in asymptomatic individuals therefore represents a key research priority.

The inferred potential for Mtb colonization (distinguished from “infection” by the absence of a detectable host immunological response36) seems an important consideration when designing anti-Mtb vaccines and/or deciding who to vaccinate, especially in TB-endemic regions. That is, the persistent bioaerosol release of Mtb within this community could indicate a homeostatic interaction between the bacillus and its human host. Although half of the participants were QFT positive, there was no correlation with aerosol Mtb exhalation, suggesting that current methods for detecting Mtb infection and estimating patient infectiousness are insufficient. Notably, HIV status was not associated with aerosolized Mtb, further implying a lack of immune interaction between host and pathogen. Our observations, together with previous studies linking the aerosol release of Mtb to infectiousness,21,22 imply a large reservoir of potential transmitters that remains invisible to national TB programs. However, the applicability of bioaerosol sampling techniques as screening tools for preventing Mtb transmission is predicated on the definitive demonstration that incident infections, and importantly, TB cases, arise from this population of relatively well individuals. Further studies are planned to establish the attributable fraction of TB transmission from this population; the immediate implications, though, are that a focus on treatment as the sole intervention to prevent transmission will require reassessing if the attributable fraction is significant in high TB-burden settings.

The observation that Mtb bioaerosol release is common in this community and is not detectable by standard measures of Mtb infection motivates for an urgent reframing of the prevailing paradigm of Mtb transmission and infection, while also emphasizing the importance of bioaerosol sampling in understanding the etiology of TB. The notion that infection with Mtb constitutes the primary determinant of TB risk is common in infectious disease research38 and has dominated thinking about new TB interventions. Our work using advanced breath aerosol collection technology challenges this assumption: in successive studies, we have detected release of Mtb-containing bioaerosols in confirmed TB patients and a majority of randomly selected community members, and we have demonstrated the time-dependent reduction (but not elimination) of Mtb bioaerosol positivity in TB clinic attendees irrespective of TB chemotherapy. These observations reinforce the axiom that although Mtb infection is necessary for TB, it is not sufficient. It appears unlikely that TB arises solely as a consequence of host,38 bacillary,39 or environmental40,41 factors. Instead, the high proportion of Mtb bioaerosol-positive individuals detected in this setting in the absence of symptoms suggests that a poorly understood combination of these elements determines disease risk.

Finally, although the significance for TB control and the attributable transmission risk from this population remains to be established, these data provide a plausible explanation for the difficulty in linking transmission chains in high-burden regions. Since the bacilli produced by randomly selected community members appear quantitatively and qualitatively indistinguishable from those released by confirmed TB patients (at least using existing tools to interrogate phenotypic and/or genetic adaptations), it seems plausible that there is some contribution to ongoing Mtb transmission. By extrapolating to the broader community, the implication appears unavoidable that there are far greater numbers of disease- and symptom-free individuals with prolonged shedding of Mtb in communal settings than those with TB disease.

Limitations of the study

The results presented earlier must be interpreted in the light of the study’s limitations, including sample size and composition, as well as the potential for the false-positive identification of Mtb. Participants were recruited from randomly selected erfs during the workday between Monday and Friday. Our sample was therefore biased to individuals both willing and able to participate in the study. However, the average age, QFT status, and HIV prevalence were as expected for the community,13 suggesting that our observations are internally reliable within Masiphumelele. It seems unlikely, therefore, that the manner of participant recruitment would significantly impact the results of this study.

Our assay for the detection of Mtb release is based on the microscopic detection of bacilli matching specific morphological characteristics, which also take up with DMN-tre.28 This technique is potentially subject to the false-positive identification of Mtb, given that other Actinomycetales produce the enzyme required for the probe incorporation.42 However, we have previously used the RASC for Mtb detection by colony formation and droplet-digital PCR.29,43,44 More recently, we successfully obtained whole-genome sequence data from three bioaerosol samples after 50-day culture, importantly demonstrating the presence of Mtb, with no non-tubercular mycobacteria or other Actinobacteria found.29

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Ryan Dinkele (ryan.dinkele@uct.ac.za).

Materials availability

This study did not generate new unique reagents.

Data and code availability

• All data reported in this paper will be shared by the lead contact upon request.

• This paper does not report original code.

• Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Acknowledgments

We acknowledge support of the 10.13039/501100001322 South African Medical Research Council (MRC- RFA- UFSP- 01- 2013/CCAMP ; R.W.) and the 10.13039/100000060 National Institute of Allergy and Infectious Diseases of the US NIH (R01AI147347 ; R.W.). We are grateful for the funding received through the Myco3V TB Research Unit (U19AI162584 ; R.W. & D.F.W.) as well as from the Research Council of Norway (R&D Project 309592, D.F.W.).

We would like to thank the participants of this study for enabling this research.

Author contributions

Conceptualization and design: R.D., S.G., B.P., A.M., Z.H., A.V., R.S., A.K., D.F.W., and R.W.; acquisition of data: A.M., Z.H., A.V., and R.S.; analysis and interpretation: R.D., D.F.W., and R.W.; first manuscript draft: R.D., D.F.W., and R.W.; funding acquisition: D.F.W. and R.W. All authors critically reviewed and revised the manuscript for intellectual content and approved it prior to submission.

Declaration of interests

The authors have no conflicting interests to declare.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER	
Chemicals, peptides, and recombinant proteins	
	
DMN-Trehalose	OliLux Biosciences	oliluxbio.com/products	
	
Critical commercial assays	
	
Xpert® MTB/RIF	Cepheid	GXMTB/RIF-US-10	
	
Software and algorithms	
	
R (version 4.3.3)	R	www.r-project.org/	
lme4	Douglas et al.45	cran.r-project.org/web/packages/lme4/index.html	

Experimental model and study participant details

Study population and participant recruitment

This study was conducted in Masiphumelele, a peri-urban township located south of Cape Town, South Africa. This residential area is divided into demarcated parcels of land, called “erfs” under South African legislation, each with its own unique numeric identifier. Erfs were randomly selected and all consenting participants over 14 years of age were eligible for recruitment into this study. Recruitment was conducted between February–December 2022, with ethical approval from the Human Research Ethics Committee of the University of Cape Town (HREC no. 529/2019).

The Respiratory Aerosol Sampling Chamber

The RASC is a purpose-built personal clean room equipped with a high efficiency bioaerosol collection system, which captures all exhaled particulate matter (bioaerosols) at 100–300 L/min in 15 mL of collection medium (sterilized phosphate-buffered saline supplemented with Polymyxin B, Amphotericin B, Nalidixic acid, Trimethoprim and Azilocillin (PANTA) [Becton Dickinson]). Bioaerosol samples were concentrated and stained with DMN-trehalose (Olilux Biosciences Inc.) overnight, as previously reported, before transfer to nanowell devices for imaging.28

Method details

Bioaerosol generation and sample processing

This study was conducted in two phases (Figure 1). In the first phase, participants 1–39 underwent bioaerosol collection during three respiratory maneuvers: FVC, TiBr, and induced cough, as previously described.25 During FVC and cough sampling, participants performed the designated maneuver 15 times within a 5-min period, as directed by the study nurse. For tidal breath sampling, participants were instructed to breathe normally into the bioaerosol collection system for 5 min. Each bioaerosol sample underwent independent collection, processing, and enumeration. Enumeration of Mtb bacilli was conducted by depositing concentrated and DMN-trehalose-stained bioaerosol samples onto nanowell devices. Bioaerosol positivity was determined by aggregating counts from all three maneuvers. Given the high proportion of bioaerosol positivity within this cohort, all participants were scheduled for a follow-up visit to obtain blood and sputum samples for subsequent QFT and GXP analysis.

The second phase involved the recruitment of participants 40–89 for a longitudinal examination of Mtb bioaerosols. This phase comprised 50 individuals sampled on three occasions: baseline, two weeks, and two months. At each visit, two equivalent bioaerosol samples were collected, with each involving 10 min of tidal breathing interspersed with deep breaths every 30 s. Subsequently, these samples were concentrated and arrayed on nanowell devices. Blood and sputum specimens were obtained at baseline for QFT and GXP analysis, respectively.

In all cases, microscopists were blinded to the origin of the sample (which included an empty booth negative control) and enumerated putative Mtb bacilli per sample.

QuantiFERON-TB gold assay

In this study, Bio Analytical Research Corporation South Africa (BARC SA) were contracted to perform and interpret QFT assays according to the manufacturer’s instructions (Qiagen).

Sputum collection and the GeneXpert assay

For all participants, sputum sampling was attempted. In those capable of producing sputum, a GXP assay was performed and interpreted according to the manufacturer’s instructions (Cepheid). Individuals unable to produce sputum were considered sputum-GXP negative.

Quantification and statistical analysis

Statistical methods

For the descriptive statistics, a Fisher’s Exact Test was used for the categorical variables given the relatively small sample size. To assess age, the normality and variance of the data were assessed, and a Student’s T Test was used for the comparison. Generalized linear regression was used to assess the bioaerosol results, either as a dichotomous outcome (logistic regression) or a count outcome (negative binomial regression). These outcomes were regressed against age, biological sex, previous TB, QFT results, and HIV status. For the respiratory maneuver comparison, we used the equivalent mixed effects regression models using lme445 in to account for the fact that each participant produced three samples. All statistical analyses were conducted in R version 4.3.3.46
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